The overall goal of this proposal is to support my lifetime career commitment to understanding the molecular mechanism of opioid actions and, more specifically, to search for the molecular basis of opioid tolerance. The current proposal aims to study the molecular details in tissue- (or cell-) specific regulation of opioid gene expression and to test the hypothesis that the alteration of mu-opioid receptor synthesis and regulation is related to the mechanism of morphine action and tolerance. To achieve this goal, we plan to carry out two independent but related studies: 1) to determine the regulation of mu-opioid receptor gene expression which controls the synthesis of receptors in specific cells. We hypothesize that the differences in spatial and temporal expression of mu-opioid receptor gene are caused by (or resultant of) characteristic interactions between cis- and trans-regulatory elements using in vitro and in vivo models, 2) to determine the molecular mechanism of mu-opioid receptor regulation by a chemical modification (i.e., phosphorylation) and determine its possible relationship to morphine tolerance. In these studies, we aim to elucidate the exact role of receptor phosphorylation/dephosphorylation on cellular adaptation of chronic opioid treatment (i.e., tolerance). We also plan to pinpoint the exact phosphorylation sites of cloned opioid receptors that are involved in receptor desensitization, as well as to determine the protein kinases (or other protein factors) that are involved in this process. Finally, we plan to test the in vivo significance of this receptor modification in tolerance through a gene targeting approach.